The government may own certain rights in the present invention pursuant to grants from the National Cancer Institute (CA47451) and from the National Institute for Allergy and Infectious Diseases (AI24009 and AI1588), and the United States Public Health Service.
1. Field of the Invention
The present invention is directed to methods for blocking or delaying programmed cell death, for delivery of gene therapy to specific cells, and for treatment of cancer and other tumorgenic diseases, as well as treatment of viral infections, through the potentiation of programmed cell death in tumor or viral host cells. The present invention is also directed to assays for candidate substances which can either inhibit, or potentiate programmed cell death.
2. Description of the Related Art
a. Programmed Cell Death (Apoptosis)
In the last decade there has been increasing acceptance in the scientific community of the idea that cells may actually be internally programmed to die at a certain point in their life cycle. As an active cellular mechanism programmed cell death, or apoptosis, has several important implications. First, it is clear that such an active process can provide additional means of regulating cell numbers as well as the biological activities of cells. Secondly, mutations or cellular events which potentiate apoptosis may result in premature cell death. Third, a form of cell death which is dependent on a specific active cellular mechanism can at least potentially be suppressed. Finally, an inhibition of preprogrammed cell death would be expected to lead to aberrant cell survival and could be expected to contribute to oncogenesis.
In general, apoptosis involves distinctive morphological changes including nuclear condensation and degradation of DNA to oligonucleosomal fragments. In certain circumstances it is evident that apoptosis is triggered by or is preceeded by changes in protein synthesis. Apoptosis appears to provide a very clean process for cellular destruction, in that the cells are disposed of by specific recognition and phagocytosis prior to bursting. In this manner cells can be removed from a tissue without causing damage to the surrounding cells. Thus, it can be seen that programmed cell death is crucial in a number of physiological processes, including morphological development, clonal selection in the immune system, and normal cell maturation and death in other tissue and organ systems.
It has also been demonstrated that cells can undergo apoptosis in response to environmental information. Examples include the appearance of a stimulus, such as glucocorticoid hormones for immature thymocytes, or the disappearance of a stimulus, such as interleukin-2 withdrawal from mature lymphocytes, or the removal of colony stimulating factors from hemopoietic precursors (for a review of literature see Williams, Cell, 85; 1097-1098, Jun. 28, 1991). Furthermore, it has recently been demonstrated that the response of removal to nerve growth factor from established neuronal cell cultures mimics target removal, or axiotomy, or other methods of trophic factor removal, and it has been postulated that the cellular mechanism involved in this response is a triggering of a suicide program or programmed cell death following the nerve growth factor removal. (See Johnson et al., Neurobiol. of Aging, 10: 549-552, 1989). The authors propose a "death cascade" or "death program", which envisions that trophic factor deprivation initiates the transcription of new mRNA and the subsequent translation of that mRNA into death associated proteins which act in sequence to ultimately produce "killer proteins". Such an intracellular mechanism seems to fit well with the characteristics of apoptosis discussed above, eg., death of specific cells without the release of harmful materials and without the disruption of tissue integrity. Furthermore, the authors indicate that inhibitors of macromolecular synthesis prevented the death of neurons in the absence of nerve growth factor.
Studies have been conducted to explore the possibility that tumor cells could be eliminated by artificially triggering apoptosis. The APO-1 monoclonal antibody can induce apoptosis in several transformed human B and T cell lines. The antibody binds to a surface protein and could act either by mimicking a positive death-inducing signal or by blocking the activity of a factor required for survival. Also, anti-FAS antibodies have similar effects, and the recent cloning and sequencing of the gene for the FAS antigen has shown that it is a 63 kilodalton transmembrane receptor. Itoh et al., Cell 66: 233-243 (1991).
However, it is important to note that neither APO-1 nor FAS can function exclusively as triggers for cell death. Both are cell surface receptors that may activate quite different responses under other circumstances. Moreover, these antigens are not confined to tumor cells and their effect on normal cells is certainly an important consideration, as is the possible appearance of variants that no longer display the antigens.
It has also been demonstrated that the cell death induced by a range of cytotoxic drugs, including several used in cancer therapy, has also been found to be a form of apoptosis. In fact, the failure of apoptosis in tumor cells could be of fundamental importance in contributing not only to the evasion of physiological controls on cell numbers, but also to resistance both to natural defenses and to clinical therapy.
It has also been demonstrated that expression of the bcl-2 gene can inhibit death by apoptosis. The bcl-2 gene was isolated from the breakpoint of the translocation between chromosomes 14 and 18 found in a high proportion of the most common human lymphomas, that being follicular B cell lymphomas. The translocation brings together the bcl-2 gene and immunoglobulin heavy chain locus, resulting in an aberrantly increased bcl-2 expression in B cells. Subsequently, Henderson et al. (Cell, 65: 1107-1115, 1991) demonstrated that expression of latent membrane protein 1 in cells infected by Epstein-Barr virus protected the infected B cells from programmed cell death by inducing expression of the bcl-2 gene. Sentman et al. (Cell, 67: 879-88, Nov. 29, 1991) demonstrated that expression of the bcl-2 gene can inhibit multiple forms of apoptosis but not negative selection in thymocytes, and Strasser et al. (Cell, 67: 889-899, Nov. 29, 1991) demonstrated that expression of a bcl-2 transgene inhibits T cell death and can perturb thymic selfcensorship. Clem et al. (Science, 245: 1388-1390, Nov. 29, 1991) identified a specific baculovirus gene product as being responsible for blocking apoptosis in insect cells.
b. Herpes Virus Infections and Neurovirulence
The family of herpes virus includes animal viruses of great clinical interest because they are the causative agents of many diseases. Epstein-Barr virus has been implicated in B cell lymphoma; cytomegalovirus presents the greatest infectious threat to AIDS patients; and Varicella Zoster Virus, is of great concern in certain parts of the world where chicken pox and shingles are serious health problems. A worldwide increase in the incidence of sexually transmitted herpes simplex (HSV) infection has occurred in the past decade, accompanied by an increase in neonatal herpes. Contact with active ulcerative lesions or asymptomatically excreting patients can result in transmission of the infectious agent. Transmission is by exposure to virus at mucosal surfaces and abraded skin, which permit the entry of virus and the initiation of viral replication in cells of the epidermis and dermis. In addition to clinically apparent lesions, latent infections may persist, in particular in sensory nerve cells. Various stimuli may cause reactivation of the HSV infection. Consequently, this is a difficult infection to eradicate. This scourge has largely gone unchecked due to the inadequacies of treatment modalities.
The known herpes viruses appear to share four significant biological properties:
1. All herpes viruses specify a large array of enzymes involved in nucleic acid metabolism (e.g., thymidine kinase, thymidylate synthetase, dUTPase, ribonucleotide reductase, etc.), DNA synthesis (e.g., DNA polymerase helicase, primase), and, possibly, processing of proteins (e.g., protein kinase), although the exact array of enzymes may vary somewhat from one herpesvirus to another.
2. Both the synthesis of viral DNAs and the assembly of capsids occur in the nucleus. In the case of some herpes viruses, it has been claimed that the virus may be de-enveloped and re-enveloped as it transits through the cytoplasm. Irrespective of the merits of these conclusions, envelopment of the capsids as it transits through the nuclear membrane is obligatory.
3. Production of infectious progeny virus is invariably accompanied by the irreversible destruction of the infected cell.
4. All herpes viruses examined to date are able to remain latent in their natural hosts. In cells harboring latent virus, viral genomes take the form of closed circular molecules, and only a small subset of viral genes is expressed.
Herpes viruses also vary greatly in their biologic properties. Some have a wide host-cell range, multiply efficiently, and rapidly destroy the cells that they infect (e.g., HSV-1, HSV-2, etc.). Others (e.g., EBV, HHV6) have a narrow host-cell range. The multiplication of some herpes viruses (e.g., HCMV) appears to be slow. While all herpes viruses remain latent in a specific set of cells, the exact cell in which they remain latent varies from one virus to another. For example, whereas latent HSV is recovered from sensory neurons, latent EBV is recovered from B lymphocytes. Herpes viruses differ with respect to the clinical manifestations of diseases they cause.
Herpes simplex viruses 1 and 2 (HSV-1, HSV-2), are among the most common infectious agents encountered by humans (Corey and Spear, N. Ena. J. Med., 314: 686-691, 1986). These viruses cause a broad spectrum of diseases which range from mild and nuisance infections such as recurrent herpes simplex labialis, to severe and life-threatening diseases such as herpes simplex encephalitis (HSE) of older children and adults, or the disseminated infections of neonates. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of antiviral therapy. However, despite some successful therapy, dermal and epidermal lesions recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality. Earlier diagnosis than is currently possible would improve therapeutic success. In addition, improved treatments are desperately needed.
Extrinsic assistance has been provided to infected individuals, in particular, in the form of chemicals. For example, chemical inhibition of herpes viral replication has been effected by a variety of nucleoside analogues such as acyclovir, 5-flurodeoxyuridine (FUDR), 5-iododeoxyuridine, thymine arabinoside, and the like.
Some protection has been provided in experimental animal models by polyspecific or monospecific anti-HSV antibodies, HSV-primed lymphocytes, and cloned T cells to specific viral antigens (Corey and Spear, N. Eng. J. Med., 314: 686-691, 1986). However, no satisfactory treatment has been found.
The .gamma..sub.1 34.5 gene of herpes simplex virus maps in the inverted repeat region of the genome flanking the L component of the virus. The discovery and characterization of the gene was reported in several articles (Chou and Roizman, J. Virol., 57: 629-635, 1986, and J. Virol., 64: 1014-1020, 1990; Ackermann et al., J. Virol., 58: 843-850, 1986). The key features are: (i) the gene encodes a protein of 263 amino acid in length; (ii) the protein contains Ala-Thr-Pro trimer repeat ten times in the middle of the coding sequence; (iii) the protein is basic in nature and consists of large number of Arg and Pro amino acids; (iv) the promoter of the gene maps in the a sequence of the genome which also serves several essential viral functions for the virus; (v) the cis-acting element essential for the expression of the gene .gamma..sub.1 34.5 is contained within the a sequence, in particular, the DR2 (12 base pair sequence repeated 22 times) and U.sub.b element. This type of promoter structure is unique to this gene and not shared by other viral gene promoters.
The function of the gene .gamma..sub.1 34.5 in its ability to enable the virus to replicate, multiply and spread in the central nervous system (CNS) was demonstrated by a set of recombinant viruses and by testing their abilities to cause fatal encephalitis in the mouse brain. The mutant viruses lacking the gene therefore lost their ability to multiply and spread in the CNS and eyes and therefore is non-pathogenic. See Chou et al., Science, 250: 1212-1266, 1990.
The .gamma..sub.1 34.5 gene functions by protecting the nerve cells from total protein synthesis shutoff in a manner characteristic of programmed cell death (apoptosis) in neuronal cells. The promoter appears to contain stress response elements and is transactivated by exposure to UV irradiation, viral infection, and growth factor deprivation. These data suggest that the gene .gamma..sub.1 34.5 is transactivated in the nerve cells at times of stress to prevent apoptosis.
The significance of these findings therefore lies in the fact that .gamma..sub.1 34.5 extends viability or lends protection to the nerve cells so that in this instance, the virus can replicate and spread from cell to cell--defined as neurovirulence. It also appears that the protection can be extended to other toxic agents or environmental stresses to which the cell is subjected. An important aspect about the nature of the neurons, unlike any other cells in human, is the fact that neurons in the brain, eyes or CNS do not regenerate which forms the basis of many impaired neurological diseases. Any genes or drugs that extend the life of cells from death or degeneration can be expected to have a significant impact in the area of neural degeneration.
The role of .gamma..sub.1 34.5, and anti-apoptosis factors, in infected cells is in its early stages of elucidation. Recent studies have suggested that Epstein-Barr virus enhances the survival capacity of infected cells through latent membrane protein 1(LMP1)-induced up-regulation of bcl-2. In that system it is postulated that LMP 1 induced bcl-2 up regulation gives virus infected B cells the potential to by-pass physiological selection and gain direct access to long lived memory B cell pools. However, bcl-2 expression fails to suppress apoptosis in some situations, for example upon withdrawal of interleukin-2 or interleukin-6. Moreover, the intracellular mechanism of action of bcl-2 expression remains unknown.
c. Programmed Cell Death and Disease Therapy
In light of the foregoing, it is apparent that the expression of .gamma..sub.1 34.5 in CNS cells added an extra dimension of protection to the neurons against viral infection, and naturally ocurring and stress-induced apoptosis. An appreciation of this extra dimension of protection can be utilized in novel and innovative means for control and treatment of central nervous system (CNS) disorders. Specifically, treatment of CNS degenerative diseases, including Alzheimer's disease, Parkinson's disease, Lou Gerig's disease, and others the etiology of which may be traceable to a form of apoptosis, and the treatment of which is currently very poor, could be improved significantly through the use of either the .gamma..sub.1 134.5 gene in gene therapy or the protein expressed by .gamma..sub.1 34.5 as a therapeutic agent. This is especially critical where the death of neuronal cells is involved, due to the fact that, as noted, such cells do not reproduce post-mitotically. Since a finite number of neurons are available it is crucial to have available methods and agents for their protection and maintenance. .gamma..sub.1 134.5 is also a very useful gene for assays of substances which mimic the effect of .gamma..sub.1 34.5and block stress of biologically induced programmed cell death.
Furthermore, the HSV-1 virus, appropriately modified so as to be made non-pathogenic, can serve as a vehicle for delivery of gene therapy to neurons. The HSV-1 virus is present in neurons of the sensory ganglia of 90% of the world's human population. The virus ascends into neuronal cell bodies via retrograde axonal transport, reaching the axon from the site of infection by the process of neurotropism. Once in the neuronal cell body the virus remains dormant until some form of stress induces viral replication (e.g. UV exposure, infection by a second virus, surgery or axotomy). As noted, the use of HSV-1 as a vector would necessitate construction of deletion mutants to serve as safe, non-pathogenic vectors. Such a virus would act as an excellent vector for neuronal gene therapy and its use would be an especially important development since few methods of gene therapy provide a means for delivery of a gene across the central nervous system's blood-brain barrier.
Moreover, other viruses, such as HSV-2, picornavirus, coronavirus, eunyavirus, togavirus, rahbdovirus, retrovirus or vaccinia virus, are available as vectors for .gamma..sub.1 34.5 gene therapy. As discussed with regard to the use of HSV-1 viruses, these vectors would also be altered in such a way as to render them non-pathogenic. In addition to the use of an appropriately mutated virus, implantation of transfected multipotent neural cell lines may also provide a means for delivery of the .gamma..sub.1 134.5 gene to the CNS which avoids the blood brain barrier.
In addition, use of the HSV-1 virus with a specific mutation in the .gamma..sub.1 34.5 gene provides a method of therapeutic treatment of tumorogenic diseases both in the CNS and in all other parts of the body. The ".gamma..sub.1 34.5 minus" virus can induce apoptosis and thereby cause the death of the host cell, but this virus cannot replicate and spread. Therefore, given the ability to target tumors within the CNS, the .gamma..sub.1 34.5 minus virus has proven a powerful therapeutic agent for hitherto virtually untreatable forms of CNS cancer. Furthermore, use of substances, other than a virus, which inhibit or block expression of genes with antiapoptotic effects in target tumor cells can also serve as a significant development in tumor therapy and in the treatment of herpes virus infection, as well as treatment of infection by other viruses whose neurovirulence is dependent upon an interference with the host cells' programmed cell death mechanisms.